High Glucose Induces the Loss of Retinal Pericytes Partly via NLRP3-Caspase-1-GSDMD-Mediated Pyroptosis.

Jinhua Gan,Fangyuan Xu,Li Liu,Maomao Huang,Genyin Lan

doi:10.1155/2020/4510628

Jinhua Gan, Fangyuan Xu + Show 3 more

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https://doi.org/10.1155/2020/4510628

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Abstract

Diabetic retinopathy (DR) is one of the hallmark complications of diabetes and a leading cause of vision loss in adults. Retinal pericyte death seems to be a prominent feature in the onset of DR. Pyroptosis is an inflammatory form of programmed cell death, defined as being caspase-gasdermin-D (GSDMD)-dependent. The NOD-like receptor pyrin 3 (NLRP3) inflammasome plays an important role in mediating GSDMD activation. However, the role and mechanism of pyroptosis in the loss of retinal pericytes during the pathogenesis of DR are still unclear. In the present study, we cultured primary human retinal pericytes (HRPs) in high glucose medium; caspase-3 inhibitor DEVD, caspase-1 inhibitor YVAD, or NLRP3 inhibitor glyburide was used as intervention reagents; GSDMD was overexpressed or suppressed by transfection with an expressing vector or retroviral silencing of GSDMD, respectively. Our data showed that high glucose induced NLRP3-caspase-1-GSDMD activation and pore formation in a dose- and time-dependent manner (p < 0.05) and resulted in the inflammatory cytokines IL-1β and IL-18 and lactate dehydrogenase (LDH) release from HRPs (p < 0.05), which are all signs of HRP pyroptosis. Overexpression of GSDMD facilitated high glucose-induced pyroptosis (all p < 0.05). However, these effects were blunted by synergistically treating DEVD, YVAD, and silencing GSDMD (p < 0.05). Taken together, our results firstly revealed that high glucose induced the loss of retinal pericytes partly via NLRP3-caspase-1-GSDMD-mediated pyroptosis.

Highlights

Diabetic retinopathy (DR) is a frequent retinal microvascular complication and a leading cause of vision loss in adults [1]
The GSDMD silencing efficiency was confirmed by western blotting after 12 hours, and human retinal pericytes (HRPs) were stimulated by 30 mM high glucose alone or were intervened by 50 μM DEVD or 10 μM YVAD for 48 h; the cells and culture medium were collected for Enzyme-Linked Immunosorbent Assay (ELISA) and western blotting analysis
The results showed that the GFPGSDMD-N fusion protein was overexpressed, especially in HRPs of 30 mM high glucose group (p < 0:05), and its expression was not reversed by DEVD or YVAD; interestingly, we found that fluorescence of GFP-gasdermin-N domain (GSDMD-N) fusion protein concentrates into the cell nucleus after DEVD or YVAD treatment, but the reason was unknown (Figure 3(a))

Summary

Introduction

DR is a frequent retinal microvascular complication and a leading cause of vision loss in adults [1]. The loss of retinal pericytes is one of the earliest changes associated with DR, and it has been postulated to initiate or trigger microaneurysm formation, abnormal leakage, edema, and ischemia, provoking proliferative neovascularization in the retina [2, 3]. It has been observed in diabetic patients and in animal models of DR, the cause of pericyte death remains unknown [4, 5]. NLRP3-caspase-1 signaling, and GSDMD-N was overexpressed or suppressed by adenoviral vectors or small hairpin RNA (shRNA); we observed whether high glucose induced the pore formation and pyroptosis via NLRP3-caspase-1GSDMD signaling, aiming to elucidate the role and molecular mechanism of pyroptosis in the pathogenesis of DR

Materials and Methods

Result

Discussion